高效液相色谱法（HPLC）测定饲料中麦角固醇

目的]建立一种测定饲料中麦角固醇的高效液相色谱法。方法]首先确定波长,流动相和流速等色谱条件,然后研究KOH浓度、乙醇与KOH溶液比例、皂化温度和时间对麦角固醇得率的影响,确定最佳皂化条件。结果]高效液相色谱条件为色谱柱HC-C18（4.6 mm×150.0 mm,5μm）,流动相为纯甲醇,流速1.5 ml/min,检测波长281 nm,柱温30℃,进样量10μl;最佳皂化提取条件为KOH质量分数35%,乙醇与KOH溶液加入比例20∶10（V∶V）,皂化温度85℃,皂化时间2 h;优化后标准曲线方程为Y=12.541X-7.603,R^2=0.999 69,线性范围为0.550.0 mg/ml;添加水平为2.0,4.8和7.5 mg/kg时,加标回收率为101%105%,RSD为1.6%3.8%（n=5）,最低检测限为0.625 mg/kg。结论]该检测方法快速、灵敏、准确、可靠,适合饲料中麦角固醇的测定。

Detection of Ergosterol in Feed Based on High Performance Liquid Chromatography

Objective]To develop a high performance liquid chromatography method for the quantitative determination of ergosterol in feed.Method] After the optimum HPLC conditions including wavelength,mobile phase and flow rate were determined,the effects of saponification conditions including the concentration of KOH,the ratio of ethanol to KOH solution,saponification temperature and time on the yield of ergosterol were studied.Result] The results showed that the optimum HPLC condition were as follows: Agilent HC-C18 column(4.6 mm ×150 mm,5 μm),mobile phase pure methanol,flow rate 1.5 ml/min,detection wavelength 281 nm,column temperature 30 ℃,and the injection volume 10 μl;the optimum saponification extraction conditions were as follows: KOH concentration 35%,ethanol to KOH solution ratio 20∶10(V∶V),saponification temperature 85 ℃,saponification time 2 h;Optimized standard curve equation Y=12.541X-7.603,R2=0.999 69,the linear range was 0.5-50.0 mg/ml;the recoveries was 101%-105% and the relative standard deviation was 1.6%-3.8%(n=5) in the spiked levels at 2.0,4.8 and 7.5 mg/kg,the detection limit of melamine was 0.625 mg/kg.Conclusion] The method is a simple,sensitive and accurate method,and suitable for the quantitative detection of ergosterol in feed.